The overall goal of this project is to develop apelin (APJ) receptor agonists to treat diet-induced obesity (DIO) and related metabolic syndrome. Metabolic syndrome can lead to several life threatening conditions including hypertension, heart disease, type II diabetes, steatohepatitis, stroke, and certain types of cancer. Available medications to treat obesity have serious side effects and limited efficacy. Thus, new medications around novel targets are needed. The APJ receptor is a G-protein coupled receptor (GPCR) protein that is activated by apelin peptides and a newly described hormone called TODDLER/ELABELA. It is an emerging multi-modal target for metabolic syndrome that needs pharmacological validation. Apelin is an insulin sensitizer that promotes fatty acid oxidation. Apelin knockout (ko) mice have higher insulin levels and glucose intolerance along with increased abdominal fat and higher bodyweight. Apelin through its interaction with APJ can inhibit maturation of pre-adipocytes and lipolysis in mature adipocytes. Activation of APJ by apelin in the endothelium leads to the formation of large, non-leaky lymphatic and blood vessels that restrict the transport of FA and their uptake in adipose. In agreement with these data, apelin ko mice have vessels that are more amenable to FA transport. Another report has indicated that apelin transgenic mice are resistant to DIO by virtue of increased vascular mass and mitochondrial biogenesis in skeletal muscles. Non-peptide probes of this receptor suitable for in vivo work are not available. We have identified a structurally novel agonist scaffold for further development through high-throughput screening. We propose to refine our early lead compounds to produce in vivo probes of APJ using three iterative specific aims. Through aim 1, synthesis and refinement of APJ probes will continue using medicinal chemistry approaches. Through aim 2, these compounds will be characterized using functional and radioligand displacement assays for APJ. Potent compounds will be then characterized using a battery of ADMET and pharmacokinetic assays. Through aim 3, in vivo testing of the best compounds will be performed in a chronic model of DIO in rodents. Completion of the project will pharmacologically validate APJ as a target for medications development to treat metabolic syndrome and produce advanced lead compounds for eventual clinical development.